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Crossing the intestinal epithelial cell barrier safely and reaching the blood with therapeutic levels of bioactive insulin have been the ultimate goal of oral insulin delivery. The optimum way to overcome the barrier lies in the design of an efficient high drug loading carrier, that can protect insulin from the harsh Gastrointestinal (GI) environment and enhance its uptake and transport by epithelial cells. In the present study, we developed a multi-layered insulin loading strategy on an anionic nanoliposome surface based on electrostatic interaction with chitosan. The layer-by-layer (LbL) coated nanoliposomes achieved high insulin loading (10.7% by weight) and offered superior protection with limited release in simulated gastric fluid (SGF) (about 6% in 1 h), simulated intestinal fluid (SIF) (2% in two weeks), and phosphate buffered saline (PBS) (5% in two weeks). Intracellular imaging revealed that the LbL coated liposomes …